The invention relates to peptide synthesis and in particular relates to improved activation and coupling in solid phase peptide synthesis (“SPPS”) that proceeds at higher rates, generates fewer undesired side reactions, and produces better results.
Solid phase peptide synthesis incorporates several basic steps that are repeated as additional amino acids are added to a growing peptide chain. The “solid phase” refers to resin particles to which initial amino acids—and then the growing peptide chains—are at attached. Because the chains are attached to particles, the chains can be handled as if they were a collections of solid particles (particularly for washing and separation—e.g., filtration—steps), and thus making the overall process easier in many cases than pure solution synthesis.
The repeated steps of SPPS include deprotection, activation and coupling. Deprotection: before each cycle starts, the last acid on the peptide chain remains “protected;” i.e., its “amino” end is connected to a functional group that protects the acid from unwanted reactions. This “protecting group” is thus removed (the “deprotection” step) when the next amino acid is about to be added.
Activation: a compound (“activator”) is added to the reaction to produce an intermediate amino acid species that is more likely to couple to the deprotected acid on the peptide chain.
Coupling: the activated species connects to the existing peptide chain.
Carbodiimide Activation Methods
Probably the most commonly used and studied activation method for peptide synthesis is based on the use of carbodiimides. Their use in peptide synthesis dates back to 1955 where N,N′-dicyclohexylcarbodiimide (DCC) was used to facilitate amide bond formation. A carbodiimide contains two slightly basic nitrogen atoms which will react with the carboxylic acid of an amino acid derivative to form a highly reactive O-acylisourea compound as shown in FIG. 1. The formed O-acylisourea can then immediately react with an amine to form a peptide bond; i.e., the path shown horizontally in FIG. 1. Alternatively, the O-acylisourea can or be converted into other reactive species.
Some of these alternative reactions of O-acylisourea, however, promote undesirable pathways that may or may not lead to peptide bond formation, and these undesired pathways are also shown in FIG. 1. Conversion to the unreactive N-acylurea (FIG. 1, lower left) prevents coupling, while epimerization of an activated chiral amino acid can occur through oxazolone formation (lower right). A more desirable highly reactive symmetrical anhydride can be formed by using excess amino acid compared to the carbodiimide (FIG. 1, upper left). This approach, however, undesirably consumes an additional amino acid equivalent.
A significant improvement for carbodiimide activation methods occurred with the incorporation of 1-hydroxybenzotriazole (HOBt) as an additive during carbodiimide activation. HOBt quickly converts the O-acylisourea into an OBt ester (FIG. 1, upper right) that is highly reactive, but avoids undesirable N-acylisourea and oxazolone formation. It was later demonstrated that 1-Hydroxy-7-azabenzotriazole (HOAt) is an advantageous replacement for HOBt due to a neighboring group effect of the nitrogen at the 7-position [161]. Many other additives can be used in place of HOBt and HOAt such as 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, OxymaPure, ECHA), and 1-hydroxy-2,5-pyrrolidinedione (NHS) to list several common examples.
Typically, 1 equivalent of additive is used compared to the amount of amino acid and carbodiimide. A recent study suggested, however, that reducing the amount of additives to less than 1 equivalent may be useful; S. Nozaki, “Delay of coupling caused by excess additives,” J. Pept. Sci., vol. 12, pp. 147-153, 2006. The authors found that the acylation reaction could be hindered by salt formation between the amine and additive. The authors also found, however, that reducing additives to less than 1 equivalent slowed down the activation rate and slightly increased epimerization in segment couplings.
N,N′-Diisopropylcarbodiimide (DIC) has largely replaced DCC as the preferred carbodiimide for peptide activation. DCC undesirably produces a urea soluble only in TFA which in turn makes its use difficult for Fmoc chemistry. Additionally, DCC is a waxy solid that can be difficult to work with and has been reported to cause allergic reactions. Alternatively, DIC offers the advantages of improved solubility of its generated urea in organic solvents, lower incidence of reported allergic reactions, and a relatively low cost. The combination of DIC/HOBt is popular because of its low cost and minimal side reactions while routinely providing effective couplings.
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (“EDC”) represents another popular choice, and a large majority of these reactions are carried out using one or more of DCC, DIC, and EDC.
Recent analysis of benzotriazole based additives such as HOBt, HOAt, and 6-Cl-HOBt have led to their reclassification as class 1 explosives; K. Wehrstedt, P. Wandrey and D. Heitkamp, “Explosive properties of 1-hydroxybenzotriazoles,” J. Hazard Mater, vol. 126, pp. 1-7, 2005. This undesirable feature of benzotriazole additives has increased interest in developing suitable alternatives for benzotriazole additives such as Oxyma (ethyl 2-cyano-2-(hydroxyimino) acetate; first reported in 1973 (M. Itoh, “Peptides. IV. Racemization Suppression by the Use of Ethyl-2-Hydroximino-2-cyanoacetate and Its Amide,” Bull. Chem. Soc. Jpn., vol. 46, pp. 2219-2221, 1973). More recently, the explosive properties of Oxyma were tested by differential scanning calorimetry (DSC) as well as accelerating rate calorimetry (ARC) with favorable results as compared to HOBt; R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham and F. Albericio, “Oxyma: An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole-Based HOBt and HOAt with a Lower Risk of Explosion,” Chemistry, vol. 15, pp. 9394-9403, 2009.
As another potential disadvantage, the use of carbodiimide based activation methods under room temperature synthesis conditions can lead to high levels of deletions based upon both a relatively slow activation process and a more acidic coupling environment.
Onium Salt Activation
Avoiding the potential disadvantages of DIC activation has led to the more recent development of onium salt based activation methods. Onium salt based activation requires the use of a base which first deprotonates the carboxylic acid to generate a carboxylate anion which in turn reacts with the onium salt activator. Improved coupling has been demonstrated with many onium salts-O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU); 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP); (3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridinato-O)tri-1-pyrrolidinyl-phosphorus hexafluorophosphate (PyAOP); and 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), among others—compared to carbodiimide based activation under room temperature conditions.
FIG. 2 illustrates onium salt based activation pathways.
Using a Base During Carbodiimide-Type Activation
In a few reports, the presence of a base during carbodiimide based couplings has been investigated under room temperature coupling conditions. Beyermann et al (M. Beyermann, P. Henklein, A. Klose, R. Sohr and M. Bienert, “Effect of tertiary amine on the carbodiimide-mediated peptide synthesis,” Int. J. Peptide Protein Res., vol. 37, pp. 252-256, 1991) previously showed that carbodiimide based activation under room temperature conditions is impeded by the presence of a hindered amine base. This can occur from preferential protonation of the base thereby preventing protonation of the carbodiimide; which is a necessary first step in generating the O-acylisourea in carbodiimide based activation techniques. Beyermann et al also showed, however, that the same hindered amine base at 1 equivalent compared to the amino acid could enhance the coupling process at room temperature if it was added after the activation process was completed. In essence, by adding 1 equivalent of base, Beyermann was able to mimic the subsequent acylation conditions of onium salt and carbodiimide activation methods which led to similar results.
Carpino et al; L. Carpino, El-Faham and A., “The Diisopropylcarbodiimide/1-Hydroxy-7-azabenzotriazole System: Segment Coupling and Stepwise Peptide Assembly,” Tetrahedron, vol. 55, pp. 6813-6830, 1999 later showed that the presence of a significantly weaker base such as 2,4,6-trimethylpyridine (TMP) at 1 or 2 equivalents relative to the amino acid in carbodiimide based couplings can improve both the activation and coupling steps without interfering with the protonation of the carbodiimide. In the same study, Carpino et al (1999) also showed that the use of the stronger base DIEA at 3 or 4 equivalents to the amino acid was significantly more effective in the subsequent peptide acylation step than the weaker base TMP in a difficult 5-mer sequence. Carpino et al (1999) also showed (in agreement with Beyermann et al) that the presence of a strong base hinders the activation process and thus should only be present during the subsequent acylation step.
Thus, both Carpino et al (1999) and Beyermann et al teach that in some cases, room temperature carbodiimide based coupling methods can produce optimal results by using a pre-activation step followed by subsequent acylation in the presence of a strong base such as DIEA present in an amount of 1-4 equivalents relative to the activated amino acid. As a rationale, using at least 1 equivalent of base compared to the activated amino acid mimics onium salt based techniques where 2 equivalents of base are typically used. In the onium salt case, the first equivalent is required for carboxylate anion formation necessary during activation while the second equivalent is present for enhancing the subsequent acylation step.
Nevertheless, Beyermann et al notes that this method only matches the synthesis results observed with onium salts (BOP, TBTU) while Carpino (1999) does not make a direct comparison between onium salt coupling and a carbodiimide based coupling in the presence of a base. Thus, together Carpino (1999) and Beyermann et al teach that the acylation step after a carbodiimide based activation can be made to perform similarly to an acylation step after an onium salt based activation by incorporating a similar amount of base at room temperature.
The use of bases during the coupling process is, however, less than ideal because they can lead to undesirable side reactions. Collins et al (J. Collins, K. Porter, S. Singh and G. Vanier, “High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS),” Org. Lett., vol. 16, pp. 940-943, 2014) showed minimal cysteine epimerization at 90° C. under a carbodiimide based coupling method without the presence of any base. Palasek et al (S. Palasek, Z. Cox and J. Collins, “Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis,” J. Pept. Sci., vol. 13, pp. 143-148, 2007) showed that significant cysteine epimerization can occur under onium salt activation methods when DIEA and NMM are present at 2 equivalents. Furthermore the Fmoc protecting group is slowly labile to DIEA, and this lability can increase at higher temperatures leading to undesirable insertion sequences (which can be difficult to separate).
In a separate study, Perich et al (J. Perich, N. Ede, S. Eagle and A. Bray, “Synthesis of phosphopeptides by the Multipin method: Evaluation of coupling methods for the incorporation of Fmoc-Tyr(PO3Bzl,H)—OH, Fmoc-Ser(PO3Bzl,H)—OH and Fmoc-Thr(PO3Bzl,H)—OH,” Lett. Pept. Sci., vol. 6, pp. 91-97, 1999) compared DIC/HOBt (1:1) and DIC/HOBt/DIEA (1:1:1) activation systems to various onium salt methods in the room temperature synthesis of three 10mer phosphopeptides. They concluded that both carbodiimide methods are inferior to HBTU/HOBt/DIEA and HATU/HOAt/DIEA.
Linking and Cleavage
A fundamental initial step in SPPS is, of course, that of connecting (“linking”) the first amino acid to the selected polymer resin using an intermediate compound (“linker”) to do so. This initial linking step can require particular conditions. Such modified conditions are typically required for standard acid linkers that feature a hydroxyl group which must act as a nucleophile for coupling. Acetylation of alcohols is difficult and is typically facilitated by 4-dimethylaminopyridine (DMAP) which acts as an acetylation catalyst for alcohols; [X. Shangjie, I. Held, B. Kempf, H. Mayr, W. Steglich and H. Zipse, “The DMAP-Catalyzed Acetylation of Alcohols—A Mechanistic Study,” Chemistry, vol. 11, pp. 4751-4757, 2005. Exemplary acid linkers include the widely used HMPA and Wang linkers among others. In these instances, a modified carbodiimide based coupling technique has been used where a highly reactive symmetrical anhydride is generated in the absence of additives (ex. HOBt, HOAt, and Oxyma) and with 1 equivalent or less of DMAP added to facilitate coupling. DMAP should be avoided during the activation process because it tends to dramatically slow activation (as shown by Carpino et al and others). This procedure is well known and described in the literature (E. Atherton, N. L. Benoiton, E. Brown, R. Sheppard and B. J. Williams, “Racemization of Activaterd, Urethane-protected Amino-acids by p-Dimethylaminopyridine. Significance in Solid-phase Peptide Synthesis,” J.C.S. Chem. Comm., pp. 336-337, 1981; S. Wang, J. Tam, B. Wang and R. Merrifield, “Enhancement of peptide coupling reactions by 4-dimethylaminopyridine,” Int. J Peptide Protein Res., vol. 18, pp. 459-467, 1981; M. Pennington, “Procedures to Improve Difficult Couplings,” in Peptide Synthesis Protocols, Vols. Methods in Molecular Biology—vol. 35, Totowa, N.J., Humana Press, 1995, p. 10). Unfortunately, the method is known to cause extensive epimerization even at room temperature and is problematic for loading (linking) sensitive amino acid derivatives such as cysteine and histidine onto resins.
Heating, Elevated Temperatures, and Microwave Irradiation
As another factor, in recent years a heating step or a microwave irradiation step during SPPS has been extensively applied as a method to improve SPPS and amino acid coupling. Microwave irradiation or other known conventional heating methods have been used with both standard carbodiimide and onium salt coupling processes. Using elevated temperature during the coupling step, however, presents several challenges for peptide synthesis. During onium salt based activation methods epimerization of cysteine derivatives is substantially increased. This epimerization results from the presence of the base (typically DIEA, NMM) at elevated temperatures. Additionally, increased δ-lactam formation of arginine during activation has been observed and leads to major arginine deletions in certain sequences; P. White, J. Collins and Z. Cox, “Comparative study of conventional and microwave assisted synthesis,” in 19th American Peptide Symposium, San Diego, Calif., 2005.
Recently, Collins et al (J. Collins, K. Porter, S. Singh and G. Vanier, “High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS),” Org. Lett., vol. 16, pp. 940-943, 2014) showed that very rapid and efficient couplings could be performed by in-situ carbodiimide based couplings at 90° C. without any base. This demonstrated that microwave irradiation is capable of accelerating both the slow activation process and subsequent acylation step in 2 minutes at 90° C. Avoiding any base present during the Collins coupling process offered the advantages that activation was not hindered in the manner described by Carpino (1999) and Beyermann et al and that the coupling environment was safer from epimerization. In fact Collins et al showed that Fmoc-Cys(Trt)-OH could be coupled at 90° C. without an increase in epimerization compared to room temperature methods. Therefore, Collins et al (J. Collins, K. Porter, S. Singh and G. Vanier, “High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS),” Org. Lett., vol. 16, pp. 940-943, 2014) teaches that an optimal use of carbodiimide chemistry at elevated temperatures avoids the use of bases.
As a disadvantage, however, the more acidic environment at higher temperatures required to drive the less reactive carbodiimide activation (compared to onium salts) tends to lead to premature cleavage of peptides attached to hyper-acid sensitive linkers (e.g., 2-chlorotrityl). Such premature cleavage can result in total loss of peptide from the resin and can significantly limit the temperatures that can be applied with this class of linkers.
Hyper-acid sensitive linkers are, however, of major importance in peptide synthesis because they allow for peptide fragment condensation which allows for larger peptide sequences to be constructed. Bulky hyper-acid linkers (such as 2-chlorotrityl) are also uniquely important for avoiding important side reactions such as diketopiperazine formation, avoiding DMAP during resin loading, and beta-elimination of c-terminal cysteine residues connected to acid linkers.
In brief and potentially partial summary, the advantages of higher temperature or microwave-assisted SPPS are offset by several disadvantages. As one disadvantage, the combination of a DIC activator, an acidic environment, and certain resins leads to (i) early (undesired) cleavage; and (ii) slower coupling after activation.
As an alternative disadvantage, an onium activator requires at least one equivalent of base to add each acid, but the extra-base will tend to racemize some acids and will degrade others.
As a third potential disadvantage, bases affect the stability of amino acids at high temperature; a factor that reduces the reaction time window, particularly for certain acids such as arginine.
Therefore, a peptide chemist faces numerous, and sometimes competing, limitations when applying elevated temperature to the coupling step in peptide synthesis with either carbodiimide or onium salt based activation methods.